Project Summary/Abstract In the malaria parasite, gene regulation is heavily dependent on mechanisms acting at the post- transcriptional and translational levels. More importantly, Plasmodium possesses many atypical characteristics in both pathways when compared to its human host. These unusual features hence extend our therapeutic window against this deadly parasite, yet these characteristics have been poorly explored at the molecular level. The main goal of this project is to characterize the role of newly identified apicomplexan-specific RNA- binding proteins {RAPs) in parasite development and virulence, and to validate their potential as novel drug targets. The fact that Plasmodium parasites modulate part of his gene expression at the post-transcriptional level was inspired by many early studies that demonstrated that the global proteomic profiles often lagged behind the transcriptional profiles. Our newly published large-scale bioinformatics reinforce this conjecture as we determined that at least 18% of all coding-gene are predicted to be potential RNA binding domain-containing protein (RBP). Furthermore, many of these parasite RBPs were experimentally shown to interact in situ with mRNAs. In all eukaryotic organisms, RBPs are essential to regulate mRNA processing at multiple levels including splicing, transport, mRNA stability and turnover, as well as mRNA localization and translational efficiency. In the human malaria parasite, very few RBPs have been characterized. We propose to address this knowledge gap by examining the function of the parasite specific RNA-binding domain proteins abundant in Apicomplexans, or RAPs. More specifically, we will use state-of-the-art genomics, molecular, and cellular approaches to determine the role of the 21 identified RAPs in parasite development and survival. In Aim 1, we will characterize the essentiality, subcellular localization and phenotypes of RAPs in P. falciparum across different developmental stage using novel CRISPR/Cas9 approaches. In Aim 2, we will define the global RAP- related ribonucleoprotein interaction network across various parasite developmental stages by developing high-throughput sequencing technologies and pull-down strategies followed by mass spectrometry analysis. Our complementary approaches will not only identify key specific RBPs contributing to parasite development, but also uncover unique post-transcriptional networks in a eukaryotic human pathogen. By providing fundamental insights into mechanisms regulating translation in Plasmodium, this project will improve our ability to design new drugs and novel lines of defense against malaria and many other infectious disease agents.